NCT00001174 In order to better interpret the impact of genetic variation on the brain biology of bipolar disorder, we are pursuing a variety of functional genomics studies, including brain imaging, RNA-sequencing in post-mortem brain tissue, and cellular phenotyping of neurons derived from induced pluripotent stem cells. We have contributed genotypes and imaging data to the Enhancing NeuroImaging Genetics through Meta-Analysis (ENIGMA) brain imaging consortium, which brings together imaging and genetics data from around the world in order to generate the large sample sizes needed for robust results. In addition, enrollees in our ongoing genetic study of BD in people of Anabaptist ancestry (ZIA-MH002843) are referred to UMD where they undergo diffusion tensor imaging as part of the ongoing Amish Connectome Study. Neuroimaging endophenotypes may shed light on the mechanisms whereby common genetic variants influence risk for a variety of psychiatric disorders. Following upon published pilot studies, we have completed RNA-sequencing in an additional 200 postmortem brain samples obtained from people with and without major psychiatric disorders (BD, schizophrenia and major depression). This study focused on the subgenual anterior cingulate cortex, especially implicated in people with mood disorders. Gene co-expression analysis highlighted immune-related genes and pathways which are specifically expressed in microglia. Gene-level and transcript-level analyses point to a major role for alternative splicing in differentiating major psychiatric disorders. Common genetic variants implicated in risk for BD or SCZ by genome-wide association studies were significantly enriched in variants that affect gene splicing. These variants also explained a substantially greater proportion of heritability than variants affecting gene expression. The results illustrate the enormous diversity of brain-expressed transcripts, demonstrate that major psychiatric disorders can be distinguished at the transcript level, and suggest that alternative splicing is one mechanism through which differences in gene expression arise in the brain. Ongoing studies in this sample include experimental validation of differentially expressed genes and transcripts, further analysis of co-expression networks, and single-cell RNA sequencing, which will help us identify the cell-type specific genes and transcripts that are dysregulated in the brains of people with mental illness. We also seek to model the functional genomics of disease-related genes in cells derived from induced pluripotent stem cell (iPSC) lines. iPSC-derived neural progenitor cells (NPCs) provide a renewable cellular template to conduct various investigations into disease-associated features and mechanisms at the early stage of neuronal development. This project aims to explore the ways in which we can use iPSC technology to study the biological impact of genes and genetic mutations that we identify in our other ongoing studies. Working with the NIH Center for Regenerative Medicine, the National Institute of Neurological Disorders and Stroke (NINDS) and the National Heart, Lung and Blood Institute (NHLBI) stem cell cores we have so far successfully reprogrammed fibroblasts into iPSCs from 46 individuals ascertained in our ongoing family studies of bipolar disorder. We are growing the cells into neural progenitor cells, neurons, and glia. These cells are then studied with high-resolution microscopic imaging, electrophysiology, and gene expression methods. These data could reveal differences between control and patient-derived cells and the impact of known and novel therapeutic agents. In collaboration with Joseph Steiner (NINDS), we are developing a morphologic assay to measure the effect of known therapeutics and stressors on dendritic number and length following differentiation of NPCs into neurons. We are also exploring ways to measure the functional impact of genetic mutations at the cellular level and to use genome editing tools such as CRISPR-Cas9 to rescue cellular phenotypes and establish a causal role for specific genetic mutations. In the coming year, we plan to expand our iPSC lines from additional families. We also plan to apply single-cell sequencing technology to iPSC-derived cells in order to explore the diversity of gene sequence and expression across individual cells from the same donor, which can be an important source of variation in human iPSC studies. The above approaches may illuminate important features that contribute to disruption of biological mechanisms during the disease process and provide reproducible assays for high throughput screening for novel therapeutics. Multigenic disorders such as BD pose special challenges for experimental studies, since a single causative mutation is usually not identifiable. For this reason, we are also studying rare, single-gene disorders whose symptoms overlap with those seen in more common mental illnesses. Smith-Magenis syndrome (SMS) is a neurodevelopmental disorder characterized by behavioral abnormalities and disruptions in circadian rhythm. The majority of SMS cases are caused by small deletions of chromosome 17, but a few very rare cases are caused by single mutations in RAI1, a gene located within the deleted region. Study samples that carry an established single gene defect permit gene editing for comparative studies between cells with and without the causative mutation. Skin cells from people living with SMS obtained in collaboration with Ann Smith (NHGRI), the co-discoverer of SMS, have been reprogrammed into iPSCs and differentiated into neurons and other brain cells. We are using cell lines derived from three patients in order to understand the biochemical and molecular characteristics of SMS, using techniques such as qPCR, western blot, immunocytochemistry and chromatin-immunoprecipitation. A major aim is to discover if the mutated protein is being expressed in neural precursor cells, suggesting this protein may contribute to the disease, or whether the disease is mainly due to decreased expression of normal RAI1 (haploinsufficiency). Findings from this study may have relevance to other neuropsychiatric disorders with circadian rhythm disturbances, such as depression and BD. In the coming year, we intend to complete RNA-sequencing on neural cells derived from people with bipolar disorder, SMS, and unaffected relatives, both before and after treatment with therapeutic doses of lithium, valproic acid, and other medications. This will allow a first look at the impact of rare genetic variants, clinical diagnosis, and treatments on genome-wide patterns of gene expression.